The degradation of most proteins in eukaryotic cells is carried out by the ubiquitin-dependent 26S proteasome. The 26S proteasome is involved in many different cellular processes, ranging from cell-cycle regulation to antigen processing [Hershko and Ciechanover (1998) Annu. Rev. Biochem. 67:425-479]. The proteasome is composed of two large units: the 20S catalytic core complex and the 19S regulatory complex [Gerards (1998) Cell. Mol. Life Sci. 54:253-262]. The 19S complex is required for the recognition of poly-ubiquitinated protein substrates which are degraded inside the 20S complex. The barrel-shaped 20S particle is made up of four rings, each of which contains seven different subunits. The two inner rings contain β-type subunits and the outer rings comprise α-type subunits.
Two of the α-subunits of the 20S proteasome, HC8 (α7) and HC9 (α3) are known to be phosphorylated in mammalian cells. Protein kinase CK2 (casein kinase II), which is co-purified with 20S proteasome preparations, has been shown to phosphorylate HC8 at two serine residues, Ser243 and Ser250, which are localized in the C-terminus of the HC8 subunit [Mason (1996) Eur. J. Biochem. 238:453-462; Castano (1996) Biochemistry 35:3782-3789]. Recently, Bose et al. [Biochem J. (2004) 378:177-84] uncovered that treatment of cells with γ interferon resulted in the dephosphorylation of HC8 subunit and destabilization of the 26S proteasome.
The NF-κB/Rel family of transcription factors participate in inflammatory and immune cell responses, cell cycle regulation, differentiation and protection from apoptosis [Baeuerle and Baltimore, Cell 87:13-20, (1996); Ghosh, et al., Annu. Rev. Immunol. 16:225-260, (1998)]. In mammals, this family of transcription factors is comprised of five members: p65 (RelA), RelB, c-Rel, NF-κB1 (which occurs both as a precursor, p105, and in a processed form, p50) and NF-κB2 (which occurs both as a precursor, p100, and as its processed product, p52). The NF-κB protein homo- and heterodimers exist in the cytoplasm, in complex with inhibitors of the IκB family. The precursor forms of NF-κB1 and NF-κB2 (p105 and p100, respectively) contain C-terminal IκB-homologous inhibitory regions. Dimers containing these NF-κB proteins are retained in the cytoplasm by virtue of the function of the IκB-homologous regions. Moreover NF-κB1/p105 and NF-κB2/p100 can also associate with dimers of other NF-κB proteins and impose cytoplasmic retention on them. NF-κB activation occurs mainly through induced degradation of the IκB proteins or of IκB homologous regions in NF-κB1/p105 and NF-κB2/p100, and consequent translocation of the NF-κB dimers to the nucleus [Ghosh and Karin, (2002) cell S81-96].
Because of its role in inflammatory and immune cell responses, cell cycle regulation, differentiation and protection from apoptosis, activation of NF-κB plays an important role in the development of different diseases such as inflammatory diseases such as rheumatoid arthritis, asthma and inflammatory bowel disease; autoimmune diseases such as systemic lupus erythematosis; or different types of cancer [Ruland, and Mak, Semin. Immunol. 15:177-83, (2003)]. Thus, considerable efforts have been made towards uncovering agents capable of modulating NF-κB activity for use in treating NF-κB related diseases. Accordingly, a large number of molecules with immunosuppressive and anti-inflammatory properties have been studied as inhibitors of NF-κB. These include glucocorticoids and other steroid hormones, cyclosporine A, FK506, rapamycin, salicylates and gold compounds. However, the majority of these compounds have broad range of activities which often result in sever immunosuppression and other severe adverse effects.
The present inventors have previously shown that NF-κB inducing kinase (NIK) plays a central role in NF-κB activation via the canonical and alternative pathways (Ramakrishnan et al., Receptor-specific signaling for both the alternative and the canonical NF-κB activation pathways by NF-κB-inducing kinase (NIK), Immunity 21: 477-489, 2004).
While reducing the present invention to practice, the present inventors unexpectedly uncovered that NIK binds the 26S proteasomal subunit HC8 thereby activating phosphorylation of the latter. Thus, the present inventors propose that agent capable of modulating the NIK-HC8 interaction can be used to activate or suppress proteasome activity and/or NIK degradation and as a result regulate NF-κB levels thereby facilitating treatment of diseases which are associated with NF-κB activity.